InteractiveFly: GeneBrief

erect wing: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - erect wing

Synonyms -

Cytological map position - 1A8

Function - Transcription factor

Keyword(s) - neural, muscle

Symbol - ewg

FlyBase ID:FBgn0005427

Genetic map position - 1-0.0.

Classification - novel

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Rai, M., Katti, P. and Nongthomba, U. (2016). Spatio-temporal coordination of cell cycle exit, fusion and differentiation of adult muscle precursors by Drosophila Erect wing (Ewg). Mech Dev [Epub ahead of print]. PubMed ID: 27039019
Summary:
The mechanisms of cell cycle exit by myoblasts during skeletal muscle development are poorly understood. Cell cycle arrest is known to be a prerequisite for myoblast fusion and subsequent differentiation. Despite tremendous knowledge on myoblast fusion and differentiation, tissue-specific factors that spatio-temporally regulate the cell cycle exit are not well known. This study shows that the transcriptional factor/co-activator "Erect wing" (Ewg) synchronises myoblast cell cycle exit with that of the fusion process. Ewg-null myoblasts show delayed temporal development of dorsal longitudinal muscles (DLMs), a group of indirect flight muscles (IFMs), which culminates to abnormal and asymmetric muscle pattern. A role for Ewg in cell cycle exit at G1/S stage is also shown. Reducing Cyclin E dose in Ewg-null mutant rescues the lack of IFMs and flight ability. Thus, Ewg repression of Cyclin E expression is required for the arrest of myoblast proliferation and initiation of myoblast fusion and terminal differentiation.
BIOLOGICAL OVERVIEW

EWG is a novel transcription factor with homology to DNA binding proteins found in organisms as diverse as sea urchins and mammals. Mutants exhibit both neural defects (apparent during embryonic development), and muscle defects, that are noticeable during the pupal phase (DeSimone, 1995).

Each type of defect, neural or muscle, has its own developmental basis. The neural defect is due to expression of ewg in neurons, while the muscle defect is due to expression of ewg in myoblasts. In fact, ewg is the first regulatory gene to be identified in Drosophila that is expressed in imaginal myoblasts. Before discussing the muscle defects, the embryonic origin of indirect flight muscles will be examined.

Myoblasts that contribute to the indirect flight muscles (IFMs) are derived from the embryonic mesoderm and attach to imaginal discs and nerves during larval development. At the onset of metamorphosis, these myoblasts migrate over the developing adult epidermis and fuse, forming the adult muscles. One group of indirect flight muscles, the dorsal longitudinal muscles (DLMs) uses modified larval muscles as templates for their development, while another group of very similar IFMs, the dorsoventral muscles (DVMs) appears to develop by the de novo fusion of myoblasts. The innervation of the IFMs develops from the modification of larval nerves. Neurons that innervate larval muscles withdraw their termini at the onset of metamorphosis, undergo specific modifications, and send out processes that grow over the developing IFMs (DeSimone, 1995 and references).

EWG expression in myoblasts is not detected during the third larval instar, however, at the same stage, EWG is detected in all larval neurons. After the onset of metamorphosis, myoblasts associated with the wing imaginal discs migrate over the developing adult epidermis. At this stage, and until 10 hours after pupal formation (APF) EWG protein is not detected in the myoblasts. At 10 hours APF, EWG protein is detected in a small population of cells in the region where DLMs are known to develop. These EWG-positive cells overlie the larval templates that are used for the development of DLMs. By 13 hours, the staining is stronger and more cells are labeled, while by 16 hours, the alignment of labeled cells along the surface of the larval templates is a noticeable feature. EWG expression is also seen in myoblasts that will contribute to the DVMS, in the progenitors of the jump muscles and their developing fibers. The first signs of defects in IMFs in ewg mutants are observed at about 18 hours APF when degeneration of the ventral-most DLMs becomes apparent. A little later, at 20 hours, the DLMs have completely degenerated (DeSimone, 1995).

To test whether neural or myoblast expression of ewg accounts for the defect in flight muscles, ewg was expressed in neurons of ewg mutants using an elav promoter. Neural expression of ewg rescues the embryonic lethality phenotype of ewg mutation but fails to rescue the IFM defects (DeSimone, 1995) It is concluded that neural expression alone is insufficient to rescue the muscle defect.

When a heat shock ewg expression vector is introduced in ewg mutant myoblast null strains, partial restoration of muscles is obtained. It is concluded that ubiquitous ewg expression allows for myogenesis, implying that muscle expression, but not neural expression (see above) cures the muscle defect in ewg mutants. This partial rescue results in animals with three fused DLMs instead of the normal six. The innervation to each of the three 'unsplit' muscles, which do not degenerate, resembles a composite of that normally seen over the pairs of muscles that will form in the wild type. Upon expression of EWG in the developing muscles, the axons branch in a manner similar to the wild type, suggesting that at least some cues for axon branching must come from the muscle targets. In neural ewg expression, innervation prior to muscle degeneration is only partial, that is to say, innervation is incomplete. Thus at least some aspect of innervation is directed by expression of ewg in muscle (DeSimone, 1995).

Erect wing regulates synaptic growth in Drosophila by integration of multiple signaling pathways

Formation of synaptic connections is a dynamic and highly regulated process. Little is known about the gene networks that regulate synaptic growth and how they balance stimulatory and restrictive signals. This study shows that the neuronally expressed transcription factor gene erect wing (ewg) is a major target of the RNA binding protein ELAV and that EWG restricts synaptic growth at neuromuscular junctions. Using a functional genomics approach it was demonstrated that EWG acts primarily through increasing mRNA levels of genes involved in transcriptional and post-transcriptional regulation of gene expression, while genes at the end of the regulatory expression hierarchy (effector genes) represent only a minor portion, indicating an extensive regulatory network. Among EWG-regulated genes are components of Wingless and Notch signaling pathways. In a clonal analysis it was demonstrated that EWG genetically interacts with Wingless and Notch, and also with TGF-β and AP-1 pathways in the regulation of synaptic growth. These results show that EWG restricts synaptic growth by integrating multiple cellular signaling pathways into an extensive regulatory gene expression network (Haussmann, 2008).

Several pathways have been identified that stimulate synaptic growth at NMJs of Drosophila larvae (Wnt/Wingless, TGF-β/BMP and jun kinase). Overexpression of AP-1 and mutants in regulatory genes involved in Wnt/Wingless and TGF-β/BMP pathways (spinster, highwire, shaggy and the proteasome) can increase bouton numbers, suggesting that synaptic growth is regulated through the balance of stimulatory and restrictive signals. This study has identified such a restrictive role for the transcription factor EWG and, through the analysis of EWG-regulated genes, for the N pathway in the regulation of synaptic growth. Using genetic mosaics, it was further demonstrated that EWG's role in synaptic growth regulation is cell-autonomous, suggesting that the transcriptional regulator EWG mediates this restrictive effect through the alteration of transcription pre-synaptically (Haussmann, 2008).

Analysis of genes differentially expressed in ewgl1mutants revealed a rather unexpected set of genes involved in synaptic growth regulation, besides an expected number of metabolic genes due to homology of EWG to human NRF-1. Most genes that could account for the phenotype of ewgl1mutants, and that are thus expressed in the nervous system, are involved in transcriptional and post-transcriptional regulation of gene expression. Although changes of transcript levels in ewgl1mutants were mostly moderate, their significance was validated through mRNA profiling with rescued ewgl1mutants under the same conditions of RNA preparation and microarray hybridization. In addition, differences in gene expression in ewgl1mutants were validated using quantitative RT-PCR and biochemical assays with regard to predicted changes in glycogen levels based on differential regulation of genes involved in gluconeogenesis. Furthermore, genetic interaction experiments in double mutants with increased bouton numbers support that these co-regulated genes are functionally connected in regulating synaptic growth (Haussmann, 2008).

The group of neuronal genes among those differentially regulated in ewgl1mutants that have been demonstrated to have roles in synaptic growth or could account for it, is remarkably small. In particular, from the large number of cell adhesion molecules and cytoskeletal proteins present in the Drosophila genome only a handful is differentially regulated. Similar results have also been obtained in response to JNK and AP-1 signaling. These results are in contrast to changes in gene expression induced by acute or chronically enhanced neuronal activity in Drosophila seizure mutants, which also result in synaptic overgrowth. Here, the vast majority of differentially regulated genes are for cell adhesion molecules and cytoskeletal proteins or their regulators, and genes involved in synaptic transmission and neuronal excitability; transcriptional or post-transcriptional regulators comprise only a minor portion. These differences could be explained by separate pathways regulating growth independent of neuronal activity (Haussmann, 2008).

Particularly striking is the large number of genes involved in RNA processing among genes differentially expressed in ewgl1mutants. Although local regulation of gene expression is required in growth cones of navigating axons, a prominent role for pre-synaptic regulation of gene expression at the RNA level is only just emerging, but is a hallmark of post-synaptic plasticity. Several RNA binding proteins have been implicated in memory storage . osk and CPSF (cleavage and polyadenylation specificity factor) are among the genes differentially regulated in ewgl1mutants. Other genes involved in RNA processing differentially regulated in ewgl1mutants comprise the whole spectrum of regulation at the post-transcriptional level, from nuclear organization (otefin), alternative pre-mRNA processing (Pinin, CPSF, Rox8) and export/import (Segregetion distorter, Nxf2, CG11092, Karyopherin, Transportin) to transport, localization and translation (oskar, swallow, ribosomal protein genes S5 and Rpl24), and likely also include the regulation of mRNA stability (Rox8) (Haussmann, 2008).

An intriguing connection between ewg and signaling pathways involved in regulating synaptic growth is indicated by differentially regulated components of the Wg and N pathways (gro and Hairless) in ewgl1mutants. Consistent with a role of the co-repressor gro in Wg and N mediated transcriptional regulation of synaptic growth, Wg and N signaling pathways do not operate independently of ewg in genetic interaction experiments. The transcriptional regulatory networks of EWG, Wg and N seem to be highly interwoven. Overexpression of pan, the transcriptional mediator of canonical Wg signaling, which is repressed by gro, does not lead to a further expansion of synaptic growth in ewg mutants, suggesting a requirement for ewg-regulated genes. This effect could be mediated by deregulated N signaling, which is also repressed by gro, but antagonistic to Wg in synaptic growth. Thus, removal of gro, as in ewg, will relieve the repressive effect of N and antagonize the stimulatory effect of pan. In the complementary situation, removal of N increased bouton numbers further in the absence of EWG, which is consistent with an increase in Wg signaling as a result of down-regulated gro in ewg mutants. Antagonism between N and Wg pathways has also been found in wing discs, where N inhibits armadillo (arm), the transcriptional co-activator of canonical Wg signaling. Intriguingly, gro has also been found to be a target of receptor tyrosine kinase signaling and, thus, can combine additional pathways with N and Wg signaling. In addition to transcriptional hierarchies, chromatin remodeling has also been implicated in synaptic plasticity. Strikingly, CG6297, a Drosophila homologue of the histone deacetylase RPD3, is differentially expressed in ewgl1mutants and physically interacts with gro (Haussmann, 2008).

How ewg exerts its effect on TGF-β signaling is less clear. A prominent regulatory step in this pathway is the regulated degradation of the SMAD co-factor Medea by Highwire. Several genes involved in regulating protein stability are differentially down-regulated in ewg mutants (CG6759, CG3431, CG4973, CG7288, CG3455, CG9327 and CG9556). Lower expression levels of these genes might interfere with stabilization of Medea and explain why the effect of activated TGF-β signaling is not additive in the absence of EWG (tkvA GOF ewg LOF). Bouton numbers in wit null mutants are marginally increased in the absence of EWG, suggesting further that genes regulated by SMADs are involved in mediating synaptic overgrowth in ewgl1mutants. Potentially, ewg could also regulate TGF-β signaling through the endosomal pathway involving spinster and/or spichthyin (Haussmann, 2008).

Functionally related genes have been shown to be co-regulated, suggesting additional ELAV targets in EWG-regulated gene networks. Indeed, ELAV negatively regulates alternative splicing of the penultimate exon in armadillo (arm). Exclusion of this exon, which truncates the carboxyl terminus of arm, reduces Wg signal transduction, which is in agreement with ewg's antagonistic role relative to Wg signaling. Another known ELAV target gene is neuroglian (nrg), where a role in synapse formation has recently been demonstrated in the giant fiber system. Taken together, the establishment of a gene network regulated by EWG will now serve as valuable tool to identify further ELAV regulated modules that shape the synapse (Haussmann, 2008).

The transcription factor EWG is a major target of the RNA binding protein ELAV, which regulates EWG protein expression via a splicing mechanism. EWG is required pre-synaptically and cell-autonomously at third instar neuromuscular junctions to restrict synaptic growth, demonstrating that restrictive activities at gene expression levels are also required for synaptic growth regulation. EWG mediates regulation of synaptic growth primarily by increasing transcript levels of genes involved in transcriptional and post-transcriptional regulation of gene expression. Genes at the end of the gene expression hierarchy (effector genes) represent only a minor portion of EWG-regulated genes. Since analysis of mutants in genes differentially regulated in ewgl1mutants revealed that these genes are involved in both stimulatory and restrictive pathways of synaptic growth, and since ewg genetically interacts with a number of signaling pathways (Wingless, Notch, TGF-β and AP-1), the results suggest that synaptic growth in Drosophila is regulated by the interplay of multiple signaling pathways rather than through independent pathways (Haussmann, 2008).


REGULATION

The neuronal transcription factor erect wing regulates specification and maintenance of Drosophila R8 photoreceptor subtypes

Signaling pathways are often re-used during development in surprisingly different ways. The Hippo tumor suppressor pathway is best understood for its role in the control of growth. The pathway is also used in a very different context, in the Drosophila eye for the robust specification of R8 photoreceptor neuron subtypes, which complete their terminal differentiation by expressing light-sensing Rhodopsin (Rh) proteins. A double negative feedback loop between the Warts kinase of the Hippo pathway and the PH-domain growth regulator Melted regulates the choice between 'pale' R8 (pR8) fate defined by Rh5 expression and 'yellow' R8 (yR8) fate characterized by Rh6 expression. This study shows that the gene encoding the homolog of human Nuclear respiratory factor 1, erect wing (ewg), is autonomously required to inhibit warts expression and to promote melted expression to specify pR8 subtype fate and induce Rh5. ewg mutants express Rh6 in most R8s due to ectopic warts expression. Further, ewg is continuously required to maintain repression of Rh6 in pR8s in aging flies. This study shows that Ewg is a critical factor for the stable down-regulation of Hippo pathway activity to determine neuronal subtype fates. Neural-enriched factors, such as Ewg, may generally contribute to the contextual re-use of signaling pathways in post-mitotic neurons (Hsiao, 2013).

The proper specification of photoreceptor subtypes including Rhodopsin expression is critical for proper color-detection and related behavior. This study found that the neural-specific transcription factor Ewg contributes to R8 subtype specification. In the absence of ewg, most pR8s are mis-specified as yR8s. This analysis shows that ewg acts autonomously in R8 to regulate the Warts-Melted feedback loop controlling subtype fate. Ewg appears to regulate pR8 fate by promoting melted expression and warts repression, suggesting that ewg is necessary to promote the complete pR8 fate rather than directly regulating Rh5 expression. Furthermore, epistasis experiments with merlin, warts and melted place ewg genetically upstream of the Warts-Melted feedback loop (Hsiao, 2013).

In addition to its role in R8 subtype establishment, ewg also functions in subtype maintenance in adult flies, as Rh6 is de-repressed in pR8 and is co-expressed with Rh5 in 4-week old ewg mutant flies. Expression of Rh5 still remains in old pR8s. ewg mutants also progressively lose melted expression and gain warts expression in R8s. The gradual disappearance of melted in old ewg mutant flies likely allows expression of warts and reactivation of Rh6. This represents another genetic program required to maintain gene expression in differentiated sensory neuron subtypes of adult animals. Previous studies have shown that the Hippo pathway is required both to specify and to maintain yR8 subtypes. Removing merlin after eclosion results in de-repression of Rh5 in all yR8s and co-expression with Rh6, a phenotype opposite to that of old ewg mutant flies. Furthermore, an active Rh6 protein is required to repress Rh5 to maintain its exclusive expression in yR8s, as loss of Rh6 results in the expansion of Rh5 to all R8s in old flies. These results are consistent with the model that establishment and maintenance programs are coupled by using the same genes, resulting in efficient long-term gene regulation (Hsiao, 2013).

How does the Ewg protein function in R8 subtype specification? Ewg has the same consensus DNA binding site as Nuclear respiratory factor 1. However, no motifs were found matching the Ewg consensus sequence in the regulatory regions of melted and warts. The diverse transcriptional targets of Ewg in various organisms also prevent a clear assignment of a conserved Ewg protein function. For example, Nuclear respiratory factor 1 acts as a transcriptional activator in the regulation of expression of cytochrome C and mitochondrial genes. However, the sea urchin Ewg homolog, P3A2, limits expression of the cytoskeletal cyIIIA actin gene. As human Nuclear respiratory factor 1 functions as an activator while sea urchin P3A2 negatively regulates cyIIIA, Ewg therefore appears to act either as an activator or as a repressor, consistent with the presence of a C-terminal activation domain and an N-terminal repression domain identified in Drosophila. Although Ewg functions upstream of the warts/melted loop, neither warts nor melted contain canonical Ewg binding motifs, suggesting that Ewg likely regulates these genes indirectly (Hsiao, 2013).

Several other genes are expressed in all photoreceptors and act as permissive factors to regulate specific Rhodopsins and photoreceptor subtypes. For example, Orthodenticle (Otd), the fly homolog of vertebrate Crx and Otx proteins, is a K50 homeoprotein expressed in all photoreceptors. Loss of otd results in the loss of Rh3 and Rh5 in p ommatidia and de-repression of Rh6 in outer photoreceptors. However, like Ewg, Otd is not sufficient to activate these genes when mis-expressed. Otd is therefore a permissive factor that likely acts with co-factors to specify their activating or repressive functions in particular photoreceptors. For Rh3, restricted Rh expression is achieved by repression by Dve, Senseless and Prospero. The same principle might apply for Ewg: since Ewg is expressed in all photoreceptors, it might recruit co-factors specific to pR8 to promote the expression of melted or to negatively regulate the Hippo pathway. Recently, ewg was shown to be required for the recruitment of the cell specific Armadillo-TCF adapter, Earthbound 1 (Ebd1), to specific chromatin sites to activate a subset of Wingless target genes. Ebd1 shares similar polytene chromatin binding sites with Ewg. It is possible that Ewg recruits a specific co-factor such as Ebd1 to function in pR8. However, no decrease was observed in Rh5 expression in ebd1 mutant retinas, suggesting that Ewg acts differently in the retina. Nevertheless, it is likely that another subtype specific co-factor functions with Ewg to specify pR8 fate (Hsiao, 2013).

In conclusion, ewg is autonomously required to specify the pR8 subtype and induce Rh5 expression. ewg appears to act upstream of the Hippo pathway, of melted, and the feedback loops to determine pR8 fate. Therefore, a neuronal specific transcription factor, Ewg, contributes to the regulation of the Hippo pathway either directly or indirectly through regulation of melted to specify the fate of R8 photoreceptors (Hsiao, 2013).

Drosophila Erect wing (Ewg) controls mitochondrial fusion during muscle growth and maintenance by regulation of the Opa1-like gene

Mitochondrial biogenesis and morphological changes are associated with tissue-specific functional demand, but the factors and pathways that regulate these processes have not been completely identified. A lack of mitochondrial fusion has been implicated in various developmental and pathological defects. The spatiotemporal regulation of mitochondrial fusion in a tissue such as muscle is not well understood. This study shows in Drosophila indirect flight muscles (IFMs) that the nuclear-encoded mitochondrial inner membrane fusion gene, Optic atrophy 1-like (Opa1-like), is regulated in a spatiotemporal fashion by the transcription factor/co-activator Erect wing (Ewg). In IFMs null for Ewg, mitochondria undergo mitophagy and/or autophagy accompanied by reduced mitochondrial functioning and muscle degeneration. By following the dynamics of mitochondrial growth and shape in IFMs, it was found that mitochondria grow extensively and fuse during late pupal development to form the large tubular mitochondria. The evidence shows that Ewg expression during early IFM development is sufficient to upregulate Opa1-like, which itself is a requisite for both late pupal mitochondrial fusion and muscle maintenance. Concomitantly, by knocking down Opa1-like during early muscle development, it was shown that it is important for mitochondrial fusion, muscle differentiation and muscle organization. However, knocking down Opa1-like, after the expression window of Ewg did not cause mitochondrial or muscle defects. This study identifies a mechanism by which mitochondrial fusion is regulated spatiotemporally by Ewg through Opa1-like during IFM differentiation and growth (Rai, 2014).

Post-transcriptional Regulation

An unusual mode of tissue-enriched gene expression is documented that is primarily mediated by alternative and inefficient splicing. Posttranscriptional regulation of the Drosophila erect wing gene, which provides a vital neuronal function and is essential for the formation of certain muscles, has been analyzed. Its predominant protein product, the 116-kDa EWG protein, a putative transcriptional regulator, can provide all known erect wing-associated functions. Moreover, consistent with its function, the 116-kDa protein is highly enriched in neurons and is also observed transiently in migrating myoblasts. Thus, at the protein level, only one major polypeptide, a 116-kDa, 733-amino-acid-long polypeptide encoded by the SC3 cDNA open reading frame (ORF), was observed in immunoblot analysis, although many other cross-reacting bands were also observed. The translation start site of the SC3 ORF is an unconventional CTG codon, suggesting that translational regulation of ewg may be an important aspect of ewg regulation. Transgenes expressing the 116-kDa EWG protein provide compelling evidence that the 116-kDa protein is the major functional protein, as expression of 116-kDa protein in the neurons rescues lethality and general expression rescues both lethal and muscle phenotypes associated with ewg alleles. An antibody generated against the 116-kDa EWG protein selectively labels all neurons in the embryonic and larval stages and certain migrating myoblasts in early pupae, suggesting a distinct tissue-specific expression of the protein and possibly transcript. In contrast to the protein distribution, Erect wing transcripts are present in comparable levels in neuron-enriched heads and neuron-poor bodies of adult Drosophila. The analyses shows that erect wing transcript consists of 10 exons and is alternatively spliced and that a subset of introns are inefficiently spliced. It is also shown that the 116-kDa EWG protein-encoding splice isoform is head enriched. In contrast, fly bodies have lower levels of transcripts that can encode the 116-kDa protein and greater amounts of unprocessed Erect wing RNA. Thus, the enrichment of the 116-kDa protein in heads is ensured by tissue-specific alternative and inefficient splicing and not by transcriptional regulation. Furthermore, this regulation is biologically important, as an increased level of the 116-kDa protein outside the nervous system is lethal (Koushika, 1999).

Efficiency of alternative splice events that result in SC3-like transcript is higher in head RNA. Splice events that are crucial for the production of the functional SC3 transcript are inclusion of exons D and J and exclusion of exons I and E. Inclusion of exon D requires splicing of introns 3a and 3c instead of 3b. The 3' splice sites of both 3a and 3c and the 5' splice site of 3c diverge from the Drosophila consensus, making them likely targets for splicing regulation. Intron 3a is inefficiently spliced in both head and body RNAs, whereas 3c is inefficiently spliced in body RNA only. Since exclusion of D resulting from exon skipping is seen in both body and head RNAs, it is likely to be the default mode, with inclusion of D requiring a positive regulatory step. To what extent the inefficient splicing of 3a and 3c affects this regulation is difficult to assess. SC3 transcript also requires the appropriate choice of a 5' splice site, resulting in the excision of intron 6. The body RNA shows inefficient splicing in the intron 6 region and, among the spliced products, about equal amounts of exon I inclusion and intron 6 excision. It is likely that RNAs that retain intron 6 become polyadenylated as consensus sequences for polyadenylation exist in intron 6; in case these are used, transcripts that encode different C termini will be generated. In conclusion the results show that (1) ewg is more widely transcribed than previously recognized, and total EWG RNA levels in heads and bodies are comparable; (2) a subset of EWG introns are efficiently spliced, but another subset are inefficiently spliced and retained in poly(A)+ RNA; (3) EWG RNA in bodies has a greater representation of both unprocessed RNAs and RNAs that include two exons that are not part of the SC3 ORF (one of these new exons is not included in EWG transcripts present in heads); (4) SC3 ORF RNA is enriched in adult heads but low in the bodies and (5) modest expression of the SC3-encoded ORF in the body can be lethal. Thus, ewg, which is widely transcribed, is primarily regulated by posttranscriptional mechanisms (Koushika, 1999).

Although the Drosophila erect wing (ewg) gene is broadly transcribed in adults, an unusual posttranscriptional regulation involving alternative and inefficient splicing generates a 116-kDa Ewg protein in neurons, while protein expression elsewhere (or of other isoforms) is below detection at this stage. This posttranscriptional control is important, since broad expression of Ewg can be lethal. Elav, a neuron-specific RNA binding protein, is necessary to regulate Ewg protein expression in Elav-null eye imaginal disc clones and Elav is sufficient for Ewg expression in wing disc imaginal tissue after ectopic expression. Analysis of Ewg expression elicited from intron-containing genomic transgenes and cDNA minitransgenes in Elav-deficient eye discs shows that this regulation is dependent on the presence of ewg introns. Analyses of the ewg splicing patterns in wild-type and Elav-deficient eye imaginal discs and in wild-type and ectopic Elav-expressing wing imaginal discs, show that certain neuronal splice isoforms correspond to Elav levels. The data presented in this paper are consistent with a mechanism by which Elav increases the splicing efficiency of ewg transcripts in alternatively spliced regions rather than with a mechanism by which stability of specific splice forms is enhanced by Elav (Koushika, 2000).

The primary transcript of ewg, which has 10 exons, A to J, is alternatively spliced in two regions. Neuron-enriched heads and neuron-poor bodies have different EWG RNA splicing profiles. Heads show enrichment for a transcript encoding a 116-kDa protein, whereas bodies have lower amounts of the transcript that encodes the 116-kDa protein and greater amounts of unprocessed RNA. The head-enriched transcript encoding the 116-kDa protein results from inclusion of exon D and exclusion of exons E and I. Additionally, splicing of introns 3a, 3c, and 6 is inefficient, since these introns are retained in polyadenylated EWG RNA (Koushika, 2000).

Additionally, Elav promotes a neuron-enriched splice isoform of Drosophila armadillo transcript. The neuron-specific arm transcript, n-arm, is generated by an alternative splice event that results from the exclusion of exon 6 of ubiquitous-arm (u-arm). The primer pair used amplifies both u-arm and n-arm transcripts; the 147-bp smaller band corresponds to n-arm, while the 244-bp band corresponds to u-arm. To test if Elav has a role in the formation of n-arm transcripts, RNA from wild-type and elav null allele (edr) eye discs, as well as from wild-type eye discs and wing discs ectopically expressing Elav were analyzed by RT-PCR. The amount of n-arm is reduced in Elav-deficient eye discs, and in the ectopically expressing wing discs expression of n-arm is clearly induced. No change was observed in the band representing u-arm splicing. In summary, the presence of n-arm is correlated with the presence of Elav in both neural and nonneural tissues, implying that arm transcripts are regulated by Elav. Similar data were obtained for splicing of exons VIIa and VIIb of Neuroglian transcripts (Koushika, 2000).

Elav ensures that the correct alternatively spliced protein isoforms of certain genes are generated in neurons. Currently three target genes, ewg, Nrg, and arm have been identified. Both Nrg and arm are vital genes and are broadly transcribed and ubiquitous protein isoforms are broadly expressed, but an additional isoform, encoded by an alternatively spliced transcript, is pan-neurally expressed. The significance of the neural Nrg (n-Nrg) isoform is not known, but the distinct cytoplasmic domain could be important in signaling. The n-Arm isoform differs from the ubiquitous Arm (u-Arm) isoform because it lacks the Wingless interacting domain; moreover, it preferentially interacts with DE-cadherins. Even with these differences in properties, the current evidence suggest that the u-Arm is sufficient to provide the n-Arm function. Perhaps a more detailed phenotypic analysis may reveal a specific role for n-Arm (Koushika, 2000).

ewg, also a vital gene, is broadly transcribed, but the protein product, a likely transcriptional regulator, is almost exclusively neural. In the case of ewg, it is clear that the expression of the 116-kDa protein isoform is essential for viability in the nervous system and that, when expressed in nonneural tissues, it can be lethal. These Elav-regulated genes provide insight into the regulatory role of Elav in neurons. Experiments reported here demonstrate for the first time that the prevalence of neuron-specific ewg, nrg, and arm transcripts positively correlates with Elav levels, and these results are achieved through the increased use of specific splice sites (Koushika, 2000).

ELAV inhibits 3'-end processing to promote neural splicing of ewg pre-mRNA

ELAV is a gene-specific regulator of alternative pre-mRNA processing in neurons of Drosophila. A functional in vivo binding site for ELAV in neurons is described through the development of a reporter gene system in transgenic animals in combination with in vitro binding assays. ELAV binds to erect wing (ewg) RNA 3' of a polyadenylation site in the terminal intron 6. At this polyadenylation site, ELAV inhibits 3'-end processing in vitro in a dose-dependent and sequence-specific manner, and ELAV binding is necessary in vivo to promote splicing of ewg intron 6. Further, the AAUAAA poly(A) complex recognition sequence, together with ELAV, is required to regulate neural 3' splice site choice in vivo. In addition, the use of segmentally labeled RNA substrates in UV cross-linking assays suggest that ELAV does not inhibit or redirect binding of cleavage factor dCstF64 at the regulated polyadenylation site on ewg RNA. These data indicate that binding of 3'-end processing factors, together with ELAV, can regulate alternative splicing (Soller, 2003).

Although the ewg gene is ubiquitously transcribed, a salient feature is the unusual posttranscriptional regulation of this transcription factor. The last intron 6 is only spliced in the presence of ELAV, as in neurons, or when ELAV is provided ectopically. This, in turn, leads to the expression of the major Ewg protein isoform sufficient for full rescue of viability and neuronal function. A rescue reporter transgene, tcgER, has been developed that recapitulates ELAV-mediated regulation of ewg transcripts in neurons of developing and adult Drosophila flies. ELAV binds directly to ewg RNA close to an intronic pA site and inhibits 3'-end formation at this site to promote neuronal splicing of ewg intron 6 (Soller, 2003).

Several lines of in vitro and in vivo evidence converge to identify the AU4-6 motifs 3' of pA2 in ewg intron 6 as a functional ELAV-binding site. Deletions introduced in tcgER reporter transgenes show that only ~25% of intron 6 is sufficient for ELAV-dependent regulation. Within the remaining RNA, ELAV UV cross-links in neuronal nuclear extracts to AU4-6 motif containing region pA-I, but not to the flanking sequence or the 3' UTR. In addition, EMSAs show that recombinant ELAV binds with nanomolar affinity to ewg RNA pA-I. Mutational analysis further substantiates ELAV's binding to AU4-6 motifs in vitro; U-to-C substitutions considerably reduce ELAV binding in UV cross-linking assays as well as in EMSAs. Moreover, AU4-6 motifs are necessary to inhibit cleavage of substrate RNA in in vitro cleavage/pA assays with neuronal nuclear extracts or when recombinant ELAV is added to nonneuronal extract. Finally, tcgER reporter transgenes with mutated AU4-6 motifs fail to show the neuronal processing mode of ewg intron 6, demonstrating the importance of these motifs to ELAV regulation in vivo (Soller, 2003).

The ELAV-binding site on ewg RNA consists of several AU4-6 motifs, consistent with previously reported binding preferences of ELAV/Hu proteins to AU-rich sequences. Within this site individual tandem AU4-6 motifs contribute to ELAV binding, indicating that several ELAV molecules bind to ewg RNA. Recently, Hu proteins were found to interact with each other in yeast two-hybrid assays and coimmunopreciptations, and could thus potentially form a complex on binding to target RNA. This is consistent with the current observations and might indicate that ELAV/Hu proteins associate cooperatively on target RNA to form a complex (Soller, 2003).

ELAV inhibits cleavage of ewg substrate RNA in in vitro cleavage assays in a sequence-specific and concentration-dependent manner. The inhibitory activity of ELAV resides in its ability to bind RNA. Thus, ELAV is not inhibitory via titrating any essential component. This is of particular importance, since ELAV was also found to interact with dCstF64 in nuclear extracts. Although it is not yet know if the interaction of ELAV and dCstF64 is direct, inhibition of pA by ELAV cannot be explained by sequestering pA factors (e.g., dCstF64) from binding to ewg RNA in vitro. Rather, specificity in the substrate RNA and assembly of ELAV and CstF64 on ewg RNA might play a critical role in inhibiting 3'-end processing. The results, however, argue against a role of ELAV in competing with pA site recognition by Cleavage and polyadenylation specificity factor (CPSF) and Cleavage stimulation factor 64 kilodalton subunit (CstF) (Soller, 2003).

In neurons, splicing of ewg intron 6 is achieved through inhibition of intronic 3'-end formation at pA2 and distal 3' splice site selection. By what mechanism does ELAV inhibit 3'-end processing to allow splicing? ELAV's binding in the proximity of the cleavage site could slow the recruitment of cleavage factors (CF I and CF II) and/or poly(A) polymerase (PAP) resulting in a delay of the cleavage reaction. Alternatively, execution of the cleavage reaction could involve a structural rearrangement that is affected by ELAV binding. In either case, this intermediate pA complex consisting of at least CPSF and CstF, together with ELAV, alters the timing of 3'-end processing to allow for the assembly of the splicosome to the neuronal 3' splice site of intron 6 and for splicing to proceed (Soller, 2003).

Transcription and RNA processing are coupled through the C-terminal domain of the largest subunit of RNA polymerase II (pol II). Low processivity of RNA pol II could occlude the availability of a 3' splice site and thus favor intronic 3'-end processing. The short distance of only 164 nt from the AAUAAA sequence to the 3' splice site of exon I, however, makes this an unlikely scenario. Furthermore, ELAV's ability to inhibit cleavage in vitro in a concentration- and sequence-dependent manner argues against a role in stimulating RNA pol II processivity to make the neural 3' splice site available for splicing before 3'-end processing occurred (Soller, 2003).

What drives the choice of the neural splice site in ewg intron 6? An interesting alliance between ELAV and components of the pA complex in choosing the neural 3' splice site was revealed when analyzing mutations of the AAUAAA pA complex recognition sequence (DeltapA2). In DeltapA2, inclusion of exon I can occur even in the presence of ELAV, whereas in the absence of ELAV, inclusion of exon I is the major splice product. Thus, the ability of the pA site to initiate the assembly of pA factors in the presence of ELAV is key to the tight regulation of usage of the distal 3' splice site in neurons. As a consequence, exon I is not included in wild-type neurons. In nonneuronal tissue, inclusion of exon I is observed at low frequency, because the few transcripts that escape 3'-end formation at pA2 are spliced to exon I. Thus, ELAV and factors bound to the pA2 site together block the 3' splice site of exon I (Soller, 2003).

In summary, this study shows that the RNA-binding protein ELAV can inhibit 3'-end formation without affecting recognition of the pA site by CPSF and CstF64. ELAV and components of the pA complex then direct exclusive use of the distal 3' splice site to promote the neural processing mode. Because bona fide pA sites are frequently found in introns, binding of pA complex components could contribute to localize splice sites, and, as shown in this study, can regulate alternative splicing (Soller, 2003).

ELAV multimerizes on conserved AU4-6 motifs important for ewg splicing regulation

ELAV is a gene-specific regulator of alternative pre-mRNA processing in Drosophila neurons. Since ELAV/Hu proteins preferentially bind to AU-rich regions that are generally abundant in introns and untranslated regions, it has not been clear how gene specificity is achieved. A combination of in vitro biochemical experiments together with phylogenetic comparisons and in vivo analysis of Drosophila transgenes was used to study ELAV binding to the last ewg intron and splicing regulation. In vitro binding studies of ELAV show that ELAV multimerizes on the ewg binding site and forms a defined and saturable complex. Further, sizing of the ELAV-RNA complex and a series of titration experiments indicate that ELAV forms a dodecameric complex on 135 nucleotides in the last ewg intron. Analysis of the substrate RNA requirements for ELAV binding and complex formation indicates that a series of AU4-6 motifs spread over the entire binding site are important, but not a strictly defined sequence element. The importance of AU4-6 motifs, but not spacing between them, is further supported by evolutionary conservation in several melanogaster species subgroups. Finally, using transgenes it has been demonstrated in fly neurons that ELAV-mediated regulation of ewg intron 6 splicing requires several AU4-6 motifs and that introduction of spacer sequence between conserved AU4-6 motifs has a minimal effect on splicing. Collectively, these results suggest that ELAV multimerization and binding to multiple AU4-6 motifs contribute to target RNA recognition and processing in a complex cellular environment (Soller, 2005).

Based on several lines of evidence, a model is proposed for a multimeric ELAV complex consisting of 12 ELAV molecules that associate with ewg RNA between a functional poly(A) site (pA2) and exon I in intron 6 in vitro. (1) ELAV assembles with RNA into a defined and saturable RNA-protein complex when assayed by EMSA. This association occurs in an RNA substrate-specific manner, since some RNAs do not form an ELAV-RNA complex even at a concentration of 3.2 microM, which thus clearly distinguishes the ELAV-RNA complex from an unspecific aggregation. (2) Two substrate RNAs of different size form two separable complexes, demonstrating that only one RNA is present in the final ELAV complex. (3) In size exclusion chromatography experiments under physiological salt conditions, ELAV bound to ewg RNA pA2-I results in a defined complex of about 700 kDa, and the smaller RBD60 protein yields an RNA-protein complex of appropriately reduced size of about 500 kDa, suggesting assembly of a complex in the range of 12 protein molecules. (4) In stoichiometry EMSAs the final ELAV complex forms at around a ratio of one RNA per 12 ELAV molecules. (5) Titration of ELAV against RBD60 (an N-terminal truncation mutant of ELAV) at complex-forming concentrations in EMSAs reveals 13 bands, as expected for a dodecameric complex. (6) Reducing the length of the substrate RNA does not result in ELAV complexes of intermediate size, indicating that binding of ELAV as dodecameric complex is an intrinsic property of ELAV to associate with target RNA. Although the tools to demonstrate an in vivo assembly of a dodecameric ELAV complex with target RNA in fly neurons are currently not available, circumstantial evidence supports the presence of large ELAV-RNA complexes in vivo. In the nucleus, ELAV has been found to sublocalize to sites of higher concentration in discrete dots and webs, indicating that complex formation with ewg pA2-I RNA at around 350 nM in vitro could meet in vivo conditions. Further, Hu proteins have also been shown to be present in large particles in cells and neurites (Soller, 2005).

Although ELAV shares the tetramer configuration characteristics with general heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins, binding to RNA induces a rearrangement into dimers. In addition, the ELAV complex forms on 43 to 135 nucleotides of RNA pA2-I, while a tetramer unit of general hnRNPs isolated from 40S particles binds to 200 to 240 nt, and the RNA present in the 40S particle is about 500 to 800 nt in length. The length differences of the RNAs present in the ELAV complex and general hnRNP complexes likely reflect a different packaging mode. Models for hnRNP C binding to RNA have favored a loose wrapping around the tetramer. For an ELAV-RNA complex, however, a different model might apply, since the two RRMs of Sex-lethal (Sxl) and the first two RRMs of HuD, both closely related to ELAV, were shown to cover 11 nt in cocrystallization experiments. A linear assembly of 12 ELAVs with RNA is therefore unlikely, particularly with RNAs as short as 43 nt, unless not all RRMs are in contact with RNA in such a complex. Moreover, phylogenetically conserved AU4-6 motifs contain only 5 to 7 nucleotides. A possible alternate model for the assembly of the ELAV complex might therefore include that ELAV surrounds the RNA upon binding, similar to the core of Sm proteins bound to snRNA (Soller, 2005).

The assembly of a dodecameric ELAV complex on RNA pA2-I suggests that an array of repetitive cis elements might mediate complex formation. Results from various approaches show that a series of AU4-6 motifs present between pA2 and exon I in the last ewg intron are important for ELAV complex binding. (1) Using RNA substrates with mutations in AU4-6 motif element m1, m2, or m3 demonstrated that all elements spread over 135 nt contribute to ELAV binding in vitro in UV-cross-linking assays and EMSAs. (2) UV-cross-linking assays with segmentally labeled substrate RNAs using RNA pA2-ivs further demonstrate that the ELAV complex binding site extends over about 135 nt. (3) Phylogenetic analysis of the ELAV binding site reveals evolutionary conservation of six AU4-6 motifs, suggesting that an ELAV dimer might bind per AU4-6 motif. (4) Functional importance in vivo of AU4-6 motifs is further shown in ELAV-mediated splicing of the last ewg intron, using Drosophila transgenes. Although an array of AU4-6 motifs is important for ELAV complex formation, not all AU4-6 motifs contribute equally. In particular, AU4-6 motifs in the m3 element and the polypyrimidine tract have a much higher impact on high-affinity binding than the m1 and m2 elements, both in vitro and in vivo. A similar situation has been observed in the hnRNP A1 binding site in intron 3 of human immunodeficiency virus tat transcripts, and the following model has been proposed. A few hnRNP A1 molecules bind first to the high-affinity portion of the binding site and then recruit further hnRNP A1 molecules to nucleate to a higher-order complex. A similar model might also apply to ELAV complex formation. Here, high-affinity binding of few ELAV molecules to the 3' part of the complex binding site could lead to recruitment of more ELAVs that will enhance complex formation in the presence of additional AU4-6 motifs. Alternatively, clustering of binding motifs might enhance cooperative interactions among ELAVs that then trigger formation of a stable complex upon reaching local concentrations close to the stoichiometry of the final ELAV complex at a specific target site, thereby contributing to gene-specific recognition of target RNAs (Soller, 2005).

The ewg ELAV complex binding site from the melanogaster species subgroup harbors three tandem AU4-6 motifs (in m1, m2, and m3 elements) that can be aligned. Collectively, the results presented here argue against tandem AU4-6 motifs in element m1, m2, or m3 either as individual tetramer binding sites or as overlapping binding sites for two tetramers. (1) ELAV assembles as dimers in stoichiometry EMSAs. (2) Deleting tandem AU4-6 motifs does not result in ELAV complexes of intermediate size, since no one- or two-tetramer complexes are detected as the main product in EMSAs. Rather, ELAV complex formation and its affinity for a specific substrate RNA depend on length and poly(U) content of the substrate RNA. (3) Tandem AU4-6 motifs in the m1 and m2 elements are not sufficient for complex formation in EMSAs. (4) Only six AU4-6 motifs (m1r, m2r, m3l, m3r, and two in the polypyrimidine tract) are evolutionarily conserved. The additional AU4-6 motifs present in the melanogaster species subgroup might therefore represent redundancy. This is also indicated by the minimal difference in affinity between RNA pA2-I, Delta1, and Delta12 in EMSAs, as the remaining AU4-6 motifs still suffice for almost optimal positioning of the ELAV complex in this assay (Soller, 2005).

Besides increasing binding specificity, multimerization of RNA binding proteins might also provide a mechanism to locate distant RNA-processing signals by looping out intronic sequence to bring splice sites into proximity. The organization of the ELAV complex binding site illustrates flexibility in positioning of AU4-6 motifs relative to each other, since they are not strictly conserved between different Drosophila species and introducing spacer sequences only marginally affects processing of the last ewg intron 6. Forming a complex with distant recognition sequences also offers an appealing explanation for ELAV-mediated regulation of pre-mRNA processing in the more complex situation of nrg, where a distal terminal exon is chosen in the presence of ELAV. UV-cross-linking studies of the whole nrg-regulated intron reveal four areas of binding in the vicinity of splice sites and poly(A) signals. An ELAV complex bound to these extensively spaced binding sites would exclude nonneuronal RNA-processing signals from recognition. A similar model has been proposed for autoregulation of hnRNP A1 alternative splicing. Here, hnRNP A1 binds sequences flanking both sides of the regulated exon, leading to skipping of this exon (Soller, 2005).

Functional importance of multimerization has also been indicated for hnRNP A1-regulated alternative splicing of intron 3 from human immunodeficiency virus tat transcripts. Here, multimerization of hnRNP A1 in the context of RNA secondary structure on intronic and exonic splicing silencer sequences competes with other factors for exon recognition. In the case of PTB-regulated splicing of a neural exon in c-src, a neuronal form of PTB, nPTB, is postulated to interfere with multimerization of PTB and release the blocked exon for inclusion in neurons. nPTB has also been found to interact with Nova-1 and to inhibit Nova-1-stimulated GlyRalpha2 E3A alternative splicing. In Drosophila, overexpression of the ELAV family member FNE can regulate expression of ELAV, similar to autoregulation by ELAV at endogenous levels. Since ELAV family members interact in yeast two-hybrid assays, they likely can also form heteromultimeric complexes in vivo, and multimeric binding of HuB at the c-myc 3' UTR has been indicated. In addition, many other RNA binding proteins have also been shown to engage in homo- and hetero-philic interactions (Soller, 2005).

In conclusion, multimerization of RNA binding proteins into macromolecular complexes likely is an important feature of this abundant class of proteins to localize pre-mRNA processing signals in a complex cellular environment in constitutive splicing and affect use of alternative splice and polyadenylation sites. In addition, multimerization of RNA binding proteins might also provide the combinatorial setup to posttranscriptionally coordinate the expression of functionally related genes in higher eukaryotes (Soller, 2005).

Differential activity of EWG transcription factor isoforms identifies a subset of differentially regulated genes important for synaptic growth regulation

The vast majority of genes in the human genome is alternatively spliced. The functional consequences of this type of post-transcriptional gene regulation that is particularly prominent in the brain, however, remains largely elusive. This study analyzed the role of alternative splicing in the transcription factor erect wing (ewg) in Drosophila and dissected its function through differential rescue with transgenes encoding different isoforms. Transgenes expressing the SC3 ORF isoform fully rescue viability and synaptic growth defects. In contrast, transgenes expressing the δDJ isoform, that lack exons D and J, have a lower activity as inferred from their expression levels and exert reduced rescue of viability and synaptic growth defects. By comparison of the gene expression profile of ewgl1 mutants rescued either by the SC3 ORF or the δDJ transgene, a set of genes were identified whose expression is exclusively restored by the SC3 isoform. These genes are mostly involved in regulating gene expression while a core function of EWG is indicated by the regulation of metabolic genes by both isoforms. In conclusion, it was demonstrated that differential rescue with different isoform encoding transgenes of the transcription factor EWG identifies a unique set of genes associated with synaptic growth regulation (Haussmann, 2010).

This study applied gene expression profiling on microarrays to measure the read-out from transgenes of alternatively spliced isoforms from the Drosophila erect wing gene with different rescue capacities to determine gene expression programs of EWG associated with synaptic growth regulation. In particular, it was demonstrated that synaptic growth defects of ewgl1 mutants are rescued by transgenes expressing the SC3 ORF isoform in neurons while transgenes that express the ∆DJ isoform do not, although both can rescue viability of the lethal ewgl1 null allele. Analysis of the expression programs affected by these two transgenes reveals a distinct set of genes whose expression is restored by the SC3 ORF isoform in ewgl1 mutants, but not by the ∆DJ isoform. This analysis identifies a core function of the EWG transcription factor that affects expression of genes commonly regulated by both isoforms involved in metabolic processes. In contrast, the expression profile associated with the rescue of synaptic growth by the SC3 ORF transgene most prominently consists of genes involved in the regulation of gene expression. Hence, the absence of genes coding for structural proteins in this class argues for involvement of post-transcriptional regulation that does mostly not affect steady state mRNA levels. Indeed, the largest class of genes with restored expression in ewgl1 mutants by the SC3 ORF transgene are the genes involved in RNA metabolism. These genes comprise a broad spectrum of post-transcriptional regulation such as regulation of alternative splicing (Pinin and CG5519), nuclear import/export (otefin and CG4579) and differential RNA stability (Rox8 and Edc3), and/or translational regulation also involving cytoplasmic polyadenylation (swallow, oskar, wispy and CPSF160). Regulation of gene expression by cytoplasmic polyadenylation has been demonstrated to play a role in synaptic plasticity as well as learning and memory. Additional genes with proven neuronal functions are dRapsyn (CG1909) and Scgalpha, and a number of cell adhesion molecules (CG4115, CG7227 and lambik). A role downstream of EWG of these genes is further supported by mostly broad expression in the nervous system. These results are in agreement with the analysis of mutants of genes differentially regulated in ewgl1. In this study, synaptic growth defects were also predominantly found in mutants of genes involved in regulating gene expression. Using isoforms with different activities, however, provides a higher resolution of the transcriptional response since mutants of many genes have multiple functions and complicate the analysis of later developmental stages such as third instar larvae (Haussmann, 2010).

Genes differentially regulated in ewgl1 mutants are both up- and down-regulated. This is consistent with structure-function studies of EWG in reporter gene assays showing that conserved parts of EWG contribute to both positive and negative transcriptional activities. Intriguingly, the genes predominantly affected by the SC3 ORF isoform are mostly down-regulated in ewgl1 mutants arguing that transcriptional activation is more important for synaptic growth regulation. Since this class consists mostly of genes involved in the regulation of gene expression, more complicated scenarios are possible which can be assessed by follow up studies analyzing transcriptional regulation of direct EWG targets in more detail (Haussmann, 2010).

It was noticed that transcript levels of genes differentially regulated in ewgl1 mutants change only moderately. The following two reasons could account for this observation. First, only a two fold change of the SC3 ORF isoform is sufficient to induce maximum repression of synaptic growth. Second, RNA for expression profiling was extracted from whole embryos and therefore differences in expression levels of broadly expressed genes is masked by non-neuronal expression in ewgl1 mutants found for many metabolic genes or genes involved in ubiquitous processes (Haussmann, 2010).

Key to the regulation of EWG SC3 ORF expression in neurons is splicing of the last intron. The neuronal RNA binding protein ELAV is required for splicing of this intron resulting in the expression of the SC3 ORF isoform. In the last intron, ELAV binds in the proximity of a polyadenylation site and inhibits 3' end processing which results in splicing of this intron. Furthermore, ewg is a major target of ELAV as the SC3 ORF isoform can rescue post-embryonic development and synaptic growth defects of lethal elav mutants. The elaborate regulation of expression of the SC3 ORF isoform by ELAV implicates ewg in fine tuning synaptic growth which is supported by small changes in SC3 ORF concentrations being biologically active in altering bouton numbers. How expression of proteins of the ∆DJ and ∆J isoforms is regulated, is currently not known, but likely involves relief of translational repression in both neurons and muscles (Haussmann, 2010).

Initially, it was hypothesized that the conserved motif in exon J of ewg is associated with regulating a unique set of genes associated with synaptic growth regulation. The results, however, argue for a different scenario. Absence of exon J together with exon D results in lower activity of the ∆DJ isoform in rescuing viability and synaptic growth, and in restoring expression levels of differentially regulated genes in ewgl1. These results indicate that EWG has two sets of target genes with different sensitivity to EWG concentrations. One set of these genes requires lower concentrations of EWG, mostly includes metabolic genes and is predicted to be bound with higher affinity. The other set of genes requires higher concentrations, includes mostly genes involved in regulating gene expression and is predicted to be bound with lower affinity (Haussmann, 2010).

Such a gradual response to different EWG concentrations would allow for a two step adaptation to adjust to changes occurring during neuronal plasticity. First, low levels of EWG would result in metabolic adaptations independent of affecting the number of synaptic connections. This response might be very transient and involve conditional expression of the ∆DJ isoform. It has been demonstrated for intracellularly cleaved Notch that very low levels of a transcriptional regulator can be biologically active. Second, high levels of EWG would result in metabolic adaptations and restriction of synaptic growth. Such a model is consistent with the data obtained with the ∆DJ isoform, namely rescue of viability and restoring expression of genes primarily associated with metabolic functions, but no rescue of synaptic growth (Haussmann, 2010).

In conclusion, this approach to characterize the gene expression programs associated with synaptic growth regulation by using the differential rescue capacities of alternative splice isoforms of the transcription factor EWG suggests an extensive regulatory network that coordinates synaptic growth with adaptations of neuronal metabolism in synaptic plasticity (Haussmann, 2010).

Protein Interactions

Erect Wing facilitates context-dependent Wnt/Wingless signaling by recruiting the cell-specific Armadillo-TCF adaptor Earthbound to chromatin

During metazoan development, the Wnt/Wingless signal transduction pathway is activated repetitively to direct cell proliferation, fate specification, differentiation and apoptosis. Distinct outcomes are elicited by Wnt stimulation in different cellular contexts; however, mechanisms that confer context specificity to Wnt signaling responses remain largely unknown. Starting with an unbiased forward genetic screen in Drosophila, a novel mechanism was recently uncovered by which the cell-specific co-factor Earthbound 1 (Ebd1), and its human homolog jerky, promote interaction between the Wnt pathway transcriptional co-activators β-catenin/Armadillo and TCF to facilitate context-dependent Wnt signaling responses (Benchabane, 2011). In the same genetic screen an unanticipated requirement was found for Erect Wing (Ewg), the fly homolog of the human sequence-specific DNA-binding transcriptional activator nuclear respiratory factor 1 (NRF1), in promoting contextual regulation of Wingless signaling. Ewg and Ebd1 functionally interact with the Armadillo-TCF complex and mediate the same context-dependent Wingless signaling responses. In addition, Ewg and Ebd1 have similar cell-specific expression profiles, bind to each other directly and also associate with chromatin at shared genomic sites. Furthermore, recruitment of Ebd1 to chromatin is abolished in the absence of Ewg. These findings provide in vivo evidence that recruitment of a cell-specific co-factor complex to specific chromatin sites, coupled with its ability to facilitate Armadillo-TCF interaction and transcriptional activity, promotes contextual regulation of Wnt/Wingless signaling responses (Xin, 2011).

Genome-wide expression profiling has revealed that the vast majority of Wnt target genes are context specific, and, by contrast, globally activated target genes are quite rare. Factors that confer context-dependent target gene activation in response to activation of this widely used signal transduction pathway are of fundamental clinical importance, as they provide putative targets for therapeutic intervention; however, the identity of these factors, and their mechanism of action remain poorly understood, and have posed a daunting challenge for the Wnt signaling field (Xin, 2011).

An unbiased forward genetic approach was undertaken to identify genes that promote Wingless signal transduction. The design of the genetic screen allowed identification of any gene that impacts Wingless signaling downstream, or at the level of the Apc/Axin destruction complex, which in principle could encompass a wide range of functions. Surprisingly, however, among the relatively small number of genetic complementation groups revealed in the screen, two cell-specific chromatin-associated co-factors were identified that had not been implicated in Wingless signaling previously, but directly associate with each other and probably mediate the same context-specific Wingless-dependent developmental process. The combined activities of the two factors in this complex not only result in its recruitment to discrete chromatin sites, but also promote activity of the Armadillo-TCF transcription complex. Together, these unanticipated findings uncover a novel mechanism that facilitates context-specific regulation of Wingless signaling (Xin, 2011).

It was recently discovered that the novel Drosophila CENPB domain protein Ebd1 and its human homolog Jerky contribute to Wnt/Wingless target gene activation by enhancing β-catenin-TCF complex formation and β-catenin recruitment to chromatin (Benchabane, 2011). Since Ebd1 is expressed in a cell-restricted pattern, these findings provided evidence that context-dependent responses to Wnt/Wingless stimulation are facilitated by cell-specific co-factors that interact with both β-catenin/Armadillo and TCF to enhance complex formation and activity. However, whether Ebd1 conferred context specificity solely by functioning as a cell-specific Armadillo-TCF adaptor or also by associating with distinct chromatin sites was not known (Xin, 2011).

This study provides multiple lines of evidence consistent with the model that the putative sequence-specific DNA-binding transcriptional activator Ewg is a physical and functional partner of Ebd1 in regulating context-specific Wingless signaling, and is required for chromatin recruitment of the Ewg-Ebd1 complex. Together, these studies suggest that Ebd1 and Ewg act together in the same context-specific Wingless-dependent developmental process, interact functionally with Armadillo-TCF, are expressed in similar cell-restricted patterns, associate physically with each other and are recruited to similar chromatin sites throughout the genome. Furthermore, all recruitment of Ebd1 to chromatin is abolished in the absence of Ewg. Based on these findings, a model is proposed in which context specificity in Wingless signaling is achieved in part through association of Ewg with distinct enhancer sites, and recruitment of Ebd1 to these enhancers. In addition, previous work suggested that by binding both Armadillo and TCF, Ebd1 stabilizes the Armadillo-TCF complex (Benchabane, 2011). Together, these findings support the model that Ebd1 promotes recruitment of the Armadillo-TCF complex to enhancers containing Ewg-binding sites, thereby facilitating context specificity in Wingless signal transduction. Alternatively, Ebd1 might also function in an Ewg-independent manner by solely providing an Armadillo-TCF adaptor function that stabilizes the Armadillo-TCF complex and thereby facilitates transcriptional activation of a subset of Wingless target genes requiring relatively high Armadillo-TCF levels (Xin, 2011).

The ability to visualize endogenous Ebd1 and Ewg on larval salivary gland polytene chromosomes reveals that Ewg is essential for association of Ebd1 with chromatin, and that nearly all, if not all, chromatin sites with which Ebd1 associates are also bound by Ewg. By contrast, the association of Ewg with chromatin is not dependent on Ebd1; furthermore, not all Ewg-associated chromatin sites are bound by Ebd1, which probably reflects the broader role for Ewg in muscle and neuronal development. Together, these findings suggest that Ewg is necessary but not sufficient for recruitment of Ebd1 to chromatin, and either additional factors or specific DNA sequences are important for association of Ebd1 with chromatin. In particular, the presence of CENPB-type DNA-binding domains in Ebd1 indicates that Ebd1 may bind DNA directly, and therefore the association of Ebd1 with chromatin may require not only Ewg, but also sequence-specific Ebd1 DNA-binding sites. If so, Ewg might bind and remodel specific chromatin sites, thereby allowing Ebd1 to access nearby cognate binding sites or, alternatively, physical association with Ewg may result in a conformational change in Ebd1 that enables DNA binding. Similarly, association of Ebd1 with the Armadillo-TCF complex may facilitate association of Ebd1 and/or Armadillo-TCF with distinct chromatin sites; currently, this hypothesis could not be tested in vivo, since fixation and immunostaining conditions do not detect the indirect association of endogenous Armadillo with polytene chromosomes. Nonetheless, the extensive physical and genetic interactions between Ewg, Ebd1 and Wingless pathway transcriptional components, coupled with association of Ewg-Ebd1 with distinct chromatin sites support the model that context-specific activation of Wingless target genes is facilitated by interaction of Armadillo-TCF with cell-specific co-activator complexes at specific sites in chromatin (Xin, 2011).

The findings also suggest that Ebd1-Ewg complex function is required for only a context-specific subset of the Wingless-dependent processes that direct indirect flight muscles (IFM) development. For example, Wingless secreted from larval wing imaginal disc epithelia specifies the fate of myoblasts associated with the wing disc that are important for IFM development. In addition, Wingless signaling regulates the specification of tendon cells and the position of flight muscle attachment sites. Further evidence for Wingless signaling in IFM development was revealed by hypomorphic wingless mutants, some of which display a morphologically normal external appearance, but are nonetheless flightless. Indeed, analysis of these hypomorphic wingless mutants reveals defects in late steps of muscle differentiation that result in partial loss of IFMs (Benchabane, 2011). In addition, strong genetic interactions was found between Ewg and the Armadillo-TCF complex. Together with the genetic and physical interactions between Ewg and Ebd1, as well as previous findings that Ebd1 facilitates Wingless signaling in adult muscle development (Benchabane, 2011), the genetic interaction between Ewg and TCF provides further evidence supporting the model that Ewg and Ebd1 facilitate Wingless-dependent adult flight muscle development. However, no Ewg expression is detected in IFM precursors until pupation, suggesting that Ewg does not participate in early steps of Wingless-dependent specification of IFM myoblasts or tendon cells, but instead promotes later steps in IFM growth and differentiation. Previous studies revealed that context specificity in Wnt signaling responses is facilitated by either crosstalk between signal transduction pathways or replacement of TCF with tissue-specific DNA-binding transcription co-factors that associate with β-catenin. Thus, these findings suggest a novel mechanism in which a sequence-specific DNA-binding transcription factor recruits a cell-specific Armadillo-TCF adaptor to modulate context-specific Wingless signaling responses (Xin, 2011).

Ewg activity in neuronal differentiation can be functionally replaced by its human homolog nuclear respiratory factor 1 (NRF1) (Haussmann, 2008), a sequence-specific DNA-binding transcription factor important for activation of genes required in mitochondrial biogenesis and respiration, and for regulation of histone gene expression. Functional analysis of vertebrate NRF1 during development has been hampered by early lethality, as mouse Nrf1-null embryos die near the time of uterine implantation. Fortuitously, however, inactivation of the zebrafish NRF1 homolog, not really finished (nrf), has improved understanding of NRF1 function during vertebrate development; fish nrf-null mutants survive up to 14 days of development with the primary developmental defect restricted to markedly smaller brains and retinas. The tissue-restricted nature of the fish nrf-null mutant phenotype was unexpected, given the broader expression of the fish nrf gene and the global roles for NRF1 in mitochondrial and histone function revealed by in vitro studies. However, these in vivo analyses revealed that fish Nrf, like fly Ewg, acts in specific tissue-restricted developmental processes. Of note, Wnt signaling is required for patterning of the mouse central nervous system. Similarly, Wnt signaling is also crucial for brain and eye development in zebrafish, thus raising the possibility that, like Ewg, vertebrate Nrf may also facilitate a context-specific subset of Wnt-dependent developmental processes (Xin, 2011).


DEVELOPMENTAL BIOLOGY

Embryonic

The protein is present after 6 hours of embryogenesis. The level of EWG protein increases during embryogenesis and then appears to drop dramatically in third-instar larvae. A comparision of head and body extracts shows an enrichment in head preparations (DeSimone, 1993). The protein is nuclear and does nor appear in neuropil regions, axons or neuronal cytoplasm. EWG protein is first detected in the developing CNS shortly after the onset of germ band shortening (early stage 12) [Images]. Staining is barely visible at this stage and becomes progressively stronger during germ band retraction; it does not appear to change after full germ band retraction. EWG is also detected in cells of the dorsal, lateral and ventral peripheral nervous system clusters and in the head PNS structures, as well as in the brain (Fleming, 1989). It is unlikely that EWG is present in neuroblasts, since the protein is not detected until neuroblast segregation from the epidermis has been completed. The spatial and temporal distributions of EWG are remarkable similar to those of ELAV, but EWG appears later than ELAV after the birth of a neuron (DeSimone, 1993).

Larval

The six Dorsal Longitudinal flight muscles (DLMs) of Drosophila develop from three larval muscles that persist into metamorphosis and serve as scaffolds for the formation of the adult muscle fibers. In response to experimental ablation, myoblasts that would normally fuse with the larval muscle, fuse with each other instead, to generate the adult fibers in the appropriate regions of the thorax. The development of these de novo DLMs is delayed and is reflected in the delayed expression of erect wing, a transcription factor thought to control differentiation events associated with myoblast fusion. The newly arising muscles express the appropriate adult-specific Actin isoform (88F), indicating that they have the correct muscle identity. However, there are frequent errors in the number of muscle fibers generated. Ablation of the larval scaffolds for the DLMs has revealed an underlying potential of the DLM myoblasts to initiate de novo myogenesis in a manner that resembles the mode of formation of the Dorso-Ventral Muscles (DVMs), which are the other group of indirect flight muscles. Therefore, it appears that the use of larval scaffolds is a superimposition on a commonly used mechanism of myogenesis in Drosophila. These results show that the role of the persistent larval muscles in muscle patterning involves the partitioning of DLM myoblasts; in so doing, they regulate formation of the correct number of DLM fibers (Fernandes, 1996).


EFFECTS OF MUTATION

Mutants of ewg exhibit both neural and muscle phenotypes; these include breaks in the central nervous system commissures and longitudinal tracts, aberrant intersegmental axonal projections pathways in the embryo, aberrant giant fiber position in the adult fly, missing or reduced indirect flight muscles associated with erect wing posture, and adult hypoactivity. ewg is also associated with neural defects in the retina and optic ganglia. Certain ewg mutations cause embryonic lethality (DeSimone, 1993 and references). Studies of gynandromorphs indicates that there is no compelling evidence of a strictly neural origin for the muscle defect in ewg mutants (de la Pompa, 1989).

Analysis of mutants of two gene pairs stripe and erect wing, and erect wing and vertical wings reveals that these loci exhibit a synergism. In addition, a dosage effect is apparent between ewg and sr. The ewg phenotype is similar to that of stripe These interactions suggest the existence of a functional relationship between the three loci (de la Pompa, 1989).

Most ewg lethal alleles lead to either a late embryonic or early larval lethal pase, indicating that the ewg gene product is necessary for the development of more than the dorsal longitudinal flight muscles (Fleming, 1983).


EVOLUTIONARY HOMOLOGS

Identification and characterization of Erect wing homologs

The P3A2 regulatory protein interacts with specific sites in the control region of the CyIIIa actin gene. This interaction is required to confine expression of a CyIIIa.CAT fusion to the aboral ectoderm, the embryonic territory in which CyIIIa is normally utilized. P3A2 also binds specifically to similar target sites located in the regulatory region of the SM50 gene, which is expressed only in skeletogenic mesenchyme lineages. The P3A2 factor was purified by affinity chromatography from nuclear extracts of 24 h sea urchin embryos, and partial peptide sequences were used to isolate a cDNA clone encoding the complete protein. There are no significant similarities between P3A2 and any other protein in existing sequence data bases. P3A2 thus includes a novel type of DNA-binding domain. To examine the differential utilization of P3A2 in CyIIIa and SM50 genes, the specific affinity of this protein was measured for the various target sites in the regulatory DNAs of each gene, and the core target site sequences identified. The stability of P3A2 complexes formed with SM50 target sites is 50-100 times greater than that of the complexes formed with CyIIIa target sites, though the factor binds to very similar core sequence elements. P3A2 is one of at least twelve different proteins whose interaction with CyIIIa regulatory DNA is required for correct developmental expression. It might be possible to purify most of these regulatory proteins, or any other specific DNA-binding proteins of the sea urchin embryo, by using the simple procedures described for P3A2 (Calzone, 1991).

Portions of NRF-1 are closely related to sea urchin P3A2 and the erect wing (EWG) protein of Drosophila. The region of highest sequence identity with P3A2 and EWG is in the amino-terminal half of the molecule, which was found by deletion mapping to contain the DNA-binding domain, whereas the carboxy-terminal half of NRF-1 from either protein is highly divergent. The DNA-binding domain in these molecules is unrelated to motifs found commonly in DNA-binding proteins; thus, NRF-1, P3A2, and EWG represent the founding members of a new class of highly conserved sequence-specific regulatory factors (Virbasius, 1993).

Erect wing protein contains an unusual DNA binding domain that is homologous to a novel transcription factor termed alpha-Pal. In response to growth, metabolic, and other signals, eukaryotic cells regulate protein biosynthesis through post-translational mechanisms that target the alpha subunit of eukaryotic initiation factor-2 (eIF-2 alpha). Previous efforts to study transcriptional mechanisms underlying this regulation identified alpha-Pal, a transcription factor for the eIF-2 alpha gene. To gain insight into the overall biological function of alpha-Pal, its cDNA has been cloned. Sequence analysis of the encoded protein reveals that alpha-Pal is a putative bZIP transcription factor. Surprisingly, both the protein sequence and the DNA-recognition site (TGCGCATGCGCA) of this human protein are strongly homologous to those of two evolutionarily distant developmental transcription factors: P3A2 and ewg. Since P3A2 directs territory-specific transcription of muscle genes in sea urchin embryos, and ewg apparently directs transcription of flight muscle and neuronal genes in Drosophila embryos, it is likely that alpha-Pal directs similar gene transcription during human embryogenesis. In other studies, genes containing alpha-Pal-binding sequences have been identified as those involved in cellular proliferation, or the growth-responsive metabolic pathways, energy transduction, translation, and DNA replication/repair. Such data suggest that alpha-Pal also functions to modulate the transcription of metabolic genes required for cellular growth (Efiok, 1994).

A negative regulatory factor is required for correct territory-specific gene expression in the sea urchin embryo. The skeletogenic SM50 gene of Strongylocentrotus purpuratus is regulated by this factor, called P3A1. P3A1 contains two sequence elements that belong to the Zn finger class of DNA-binding motifs, and in these regions is most closely similar to the Drosophila Hunchback factor. The P3A1 factor also binds to a similar target sequence in a second gene, CyIIIa, expressed in embryonic aboral ectoderm. Another sea urchin embryo protein factor, P3A2, footprints the same target sites in the SM50 and CyIIIa genes as does P3A1, but lacks the Zn finger sequence motifs and in amino acid sequence is almost entirely dissimilar to P3A1. A deletion analysis of P3A2 delimited the DNA-binding region, revealing that five specific amino acids in the first P3A1 finger region and four in the second P3A1 finger region are also present in equivalent positions in P3A2. The P3A1 and P3A2 factors could function as regulatory antagonists, having evolved similar target specificities from dissimilar DNA-binding domains (Hoog, 1991).

SpP3A1 and SpP3A2 are DNA-binding proteins that interact specifically with the same target sites in the regulatory domains of the Strongylocentrotus purpuratus CyIIIa gene as well as several other known genes. Both proteins are present in unfertilized eggs, and both enter the embryonic nuclei early in development, but only P3A2 remains present in nuclei at functional concentrations beyond the early gastrula stage. Combined with earlier measurements of P3A site binding at cleavage stages, these measurements show that P3A1 would be replaced by P3A2 at target sites in genes regulated by these factors (Zeller, 1995).

In response to growth, metabolic, and other signals, eukaryotic cells regulate protein biosynthesis through post-translational mechanisms that target the alpha subunit of eukaryotic initiation factor-2 (eIF-2 alpha). Previous efforts to study transcriptional mechanisms underlying this regulation have identified a novel transcription factor (alpha-Pal) for the eIF-2 alpha gene. Sequence analysis of the encoded protein reveals that alpha-Pal is a putative bZIP transcription factor. Both the protein sequence and the DNA-recognition site (TGCGCATGCGCA) of this human protein are strongly homologous to those of two evolutionarily distant developmental transcription factors, P3A2 and EWG. Since P3A2 directs territory-specific transcription of muscle genes in sea urchin embryos, and EWG apparently directs transcription of flight muscle and neuronal genes in Drosophila embryos, it is likely that alpha-Pal directs similar gene transcription during human embryogenesis. Genes containing alpha-Pal-binding sequences are involved in cellular proliferation, or the growth-responsive metabolic pathways, energy transduction, translation, and DNA replication/repair. The DNA synthesis/repair genes include human DNA polmerase alpha subunit, while cellular proliferation genes include PDGF2 and Hepatocyte growth factor-like protein. Such data suggest that alpha-Pal functions to modulate the transcription of metabolic genes required for cellular growth (Efiok, 1994).

Initiation binding receptor (IBR) is a chicken erythrocyte factor (apparent molecular mass, 70 to 73 kDa) that binds to the sequences spanning the transcription initiation site of the histone h5 gene, repressing its transcription. A variety of other cells, including transformed erythroid precursors, do not have IBR but a factor referred to as IBF (68 to 70 kDa) that recognizes the same IBR sites. IBR is a 503-amino-acid-long acidic protein which is 99.0% identical to the recently reported human NRF-1/alpha-Pal factor and highly related to the invertebrate transcription factors P3A2 and erect wing gene product. IBR and IBF are most likely identical proteins, differing in their degree of glycosylation. The factor associates as stable homodimers. The dimer is the relevant DNA-binding species. The evolutionarily conserved N-terminal half of IBR/F harbors the DNA-binding/dimerization domain (outer limits, 127 to 283), one or several casein kinase II sites (37 to 67), and a bipartite nuclear localization signal (89 to 106) which appears to be necessary for nuclear targeting. Binding site selection reveals that the alternating RCGCRYGCGY consensus constitutes high-affinity IBR/F binding sites and that the direct-repeat palindrome TGCGCATGCGCA is the optimal site. A survey of genes potentially regulated by this family of factors revealed genes involved primarily in growth-related metabolism (Gomez-Cuadrado, 1995).

NRF-1 and NRF-2 provide a link between the expression of nuclear and mitochondrial genes

Mitochondrial transcription factor A (mtTFA), the product of a nuclear gene, stimulates transcription from the two divergent mitochondrial promoters and is, most likely, the principal activator of mitochondrial gene expression in vertebrates. The proximal promoter of the human mtTFA gene is highly dependent upon recognition sites for the nuclear respiratory factors, NRF-1 and NRF-2, for activity. These factors have been previously implicated in the activation of numerous nuclear genes that contribute to mitochondrial respiratory function. The affinity-purified factors from HeLa cells specifically bind to the mtTFA NRF-1 and NRF-2 sites through guanine nucleotide contacts that are characteristic for each site. Mutations in these contacts eliminate NRF-1 and NRF-2 binding and also dramatically reduce promoter activity in transfected cells. Although both factors contribute, NRF-1 binding appears to be the major determinant of promoter function. This dependence on NRF-1 activation is confirmed by in vitro transcription using highly purified recombinant proteins that display the same binding specificities as the HeLa cell factors. The activation of the mtTFA promoter by both NRF-1 and NRF-2 therefore provides a link between the expression of nuclear and mitochondrial genes and suggests a mechanism for their coordinate regulation during organelle biogenesis (Virbasius, 1994).

Nuclear respiratory factor 1 (NRF-1) is a transcription factor that acts on nuclear genes encoding respiratory subunits and components of the mitochondrial transcription and replication machinery. The NRF-1 gene spans approximately 65 kilobases (kb) and has 11 exons and 10 introns, ranging in size from 0.8 to 15 kb. NRF-1 mRNA is expressed at very low levels in rat tissues compared with cytochrome c and, unlike cytochrome c, is most abundantly expressed in lung and testis (Gopalakrishnan, 1995).

Transcription factor nuclear respiratory factor 1 (NRF-1) was originally identified as an activator of the cytochrome c gene and subsequently found to stimulate transcription through specific sites in other nuclear genes whose products function in the mitochondria. These include subunits of the cytochrome oxidase and reductase complexes and a component of the mitochondrial DNA replication machinery. A functional recognition site for NRF-1 is present in the ATP synthase gamma-subunit gene extending the proposed respiratory role of NRF-1 to complex V. In addition, biologically active NRF-1 sites are found in genes encoding the eukaryotic translation initiation factor 2 alpha-subunit and tyrosine aminotransferase, both of which participate in the rate-limiting step of their respective pathways of protein biosynthesis and tyrosine catabolism. The recognition sites from each of these genes form identical complexes with NRF-1 as established by competition binding assays, methylation interference footprinting, and UV-induced DNA cross-linking. Cloned oligomers of each NRF-1 binding site also stimulate the activity of a truncated cytochrome c promoter in transfected cells. The NRF-1 binding activities for the various target sites copurified approximately 33,000-fold and resided in a single protein of 68 kDa. These observations further support a role for NRF-1 in the expression of nuclear respiratory genes and suggest it may help coordinate respiratory metabolism with other biosynthetic and degradative pathways (Chau, 1995).

Structure of Erect wing homologs

Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) and Nrf2 regulate ARE (antioxidant response element)-driven genes. At its N-terminal end, Nrf1 contains 155 additional amino acids that are absent from Nrf2. This 155-amino-acid polypeptide includes the N-terminal domain (NTD, amino acids 1-124) and a region (amino acids 125-155) that is part of acidic domain 1 (amino acids 125-295). Within acidic domain 1, residues 156-242 share 43% identity with the Neh2 (Nrf2-ECH homology 2) degron of Nrf2 that serves to destabilize this latter transcription factor through an interaction with Keap1 (Kelch-like ECH-associated protein 1). The function of the 155-amino-acid N-terminal polypeptide was examined in Nrf1, along with its adjacent Neh2-like subdomain. Activation of ARE-driven genes by Nrf1 was negatively controlled by the NTD (N-terminal domain) through its ability to direct Nrf1 to the endoplasmic reticulum. Ectopic expression of wild-type Nrf1 and mutants lacking either the NTD or portions of its Neh2-like subdomain into wild-type and mutant mouse embryonic fibroblasts indicates that Keap1 controls neither the activity of Nrf1 nor its subcellular distribution. Immunocytochemistry showed that whereas Nrf1 gave primarily cytoplasmic staining that is co-incident with that of an endoplasmic-reticulum marker, Nrf2 gives primarily nuclear staining. Attachment of the NTD from Nrf1 to the N-terminus of Nrf2 produces a fusion protein that is redirected from the nucleus to the endoplasmic reticulum. Although this NTD-Nrf2 fusion protein exhibits less transactivation activity than wild-type Nrf2, it is nevertheless still negatively regulated by Keap1. Thus Nrf1 and Nrf2 are targeted to different subcellular compartments and are negatively regulated by distinct mechanisms (Zhang, 2006).

Expression of antioxidant and phase 2 xenobiotic metabolizing enzyme genes is regulated through cis-acting sequences known as antioxidant response elements. Transcriptional activation through the antioxidant response elements involves members of the CNC (Cap 'n' Collar) family of basic leucine zipper proteins including Nrf1 and Nrf2. Nrf2 activity is regulated by Keap1-mediated compartmentalization in the cell. Given the structural similarities between Nrf1 and Nrf2, attempts were made to investigate whether Nrf1 activity is regulated similarly to Nrf2. Nrf1 also resides normally in the cytoplasm of cells. Cytoplasmic localization however, is independent of Keap1. Colocalization analysis using green fluorescent protein-tagged Nrf1 and subcellular fractionation of endogenous Nrf1 and fusion proteins indicate that Nrf1 is primarily a membrane-bound protein localized in the endoplasmic reticulum. Membrane targeting is mediated by the N terminus of the Nrf1 protein that contains a predicted transmembrane domain, and deletion of this domain resulted in a predominantly nuclear localization of Nrf1 that significantly increased the activation of reporter gene expression. Treatment with tunicamycin, an endoplasmic reticulum stress inducer, caused an accumulation of a smaller form of Nrf1 that correlated with detection of Nrf1 in the nucleus by biochemical fractionation and immunofluorescent analysis. These results suggest that Nrf1 is normally targeted to the endoplasmic reticulum membrane and that endoplasmic reticulum stress may play a role in modulating Nrf1 function as a transcriptional activator (W. Wang, 2006).

Mutation of Erect wing homologs

Not really finished (nrf), a larval-lethal mutation in zebrafish generated by retroviral insertion, causes specific retinal defects. Analysis of mutant retinae reveals an extensive loss of photoreceptors and their precursors around the onset of visual function. These neurons undergo apoptosis during differentiation, affecting all classes of photoreceptors, suggesting an essential nrf function for the development of all types of photoreceptors. In the mutant, some photoreceptors escape cell death, are functional, and, as judged by opsin expression, belong to at least three classes of cones and one class of rods. The protein encoded by nrf is related to Drosophila Erect wing and is a close homolog of human Nuclear Respiratory Factor 1 and avian Initiation Binding Repressor, transcriptional regulators binding the upstream consensus sequence RCGCRYGCGY. At 24 hours of development, prior to neuronal differentiation, nrf is expressed ubiquitously throughout the developing retina and central nervous system. At 48 hours of development, expression of nrf is detected in the ganglion cell layer, in the neurons of the inner nuclear layer, and in the optic nerve and optic tracts, and, at 72 hours of development, is no longer detectable by in situ hybridization. Mutants contain no detectable nrf mRNA and die within 2 weeks postfertilization as larvae with reduced brain size. On the basis of its similarity with NRF-1 and IBR, nrf is likely involved in transcriptional regulation of multiple target genes, including those that encode mitochondrial proteins, growth factor receptors and other transcription factors. This demonstrates the power of insertional mutagenesis as a means for characterizing novel genes necessary for vertebrate retinal development (Becker, 1998).

In vitro studies have implicated nuclear respiratory factor 1 (NRF-1) in the transcriptional expression of nuclear genes required for mitochondrial respiratory function, as well as for other fundamental cellular activities. This study investigated he in vivo function of NRF-1 in mammals by disrupting the gene in mice. A portion of the NRF-1 gene that encodes the nuclear localization signal and the DNA-binding and dimerization domains was replaced through homologous recombination by a beta-galactosidase-neomycin cassette. In the mutant allele, beta-galactosidase expression is under the control of the NRF-1 promoter. Embryos homozygous for NRF-1 disruption die between embryonic days 3.5 and 6.5. beta-Galactosidase staining was observed in growing oocytes and in 2. 5- and 3.5-day-old embryos, demonstrating that the NRF-1 gene is expressed during oogenesis and during early stages of embryogenesis. Moreover, the embryonic expression of NRF-1 did not result from maternal carryover. While most isolated wild-type and NRF-1+/- blastocysts can develop further in vitro, the NRF-1-/- blastocysts lack this ability despite their normal morphology. Interestingly, a fraction of the blastocysts from heterozygous matings had reduced staining intensity with rhodamine 123 and NRF-1-/- blastocysts had markedly reduced levels of mitochondrial DNA (mtDNA). The depletion of mtDNA did not coincide with nuclear DNA fragmentation, indicating that mtDNA loss was not associated with increased apoptosis. These results are consistent with a specific requirement for NRF-1 in the maintenance of mtDNA and respiratory chain function during early embryogenesis (Huo, 2001).

The Nrf1 transcription factor belongs to the CNC subfamily of basic leucine zipper proteins. Knockout of Nrf1 is lethal in mouse embryos, but nothing is known about the cell types that absolutely require its function during development. This study shows by chimera analysis that Nrf1 is essential for the hepatocyte lineage. Mouse embryonic stem cells lacking Nrf1 developed normally and contributed to most tissues in adult chimeras where Nrf1 is normally expressed. Nrf1-deficient cells contribute to fetal, but not adult, liver cells. Loss of Nrf1 function results in liver cell apoptosis in late-gestation chimeric fetuses. Fetal livers from mutant embryos exhibit increased oxidative stress and impaired expression of antioxidant genes, and primary cultures of nrf1-/- fetal hepatocytes are sensitive to tert-butyl hydroperoxide-induced cell death, suggesting that impaired antioxidant defense may be responsible for the apoptosis observed in the livers of chimeric mice. In addition, cells deficient in Nrf1 are sensitized to the cytotoxic effects of tumor necrosis factor (TNF). These results provide in vivo evidence demonstrating an essential role of Nrf1 in the survival of hepatocytes during development. The results also suggest that Nrf1 may promote cell survival by maintaining redox balance and protecting embryonic hepatocytes from TNF-mediated apoptosis during development (Chen, 2003).

Nrf1 and Nrf2 are members of the CNC family of bZIP transcription factors that exhibit structural similarities, and they are co-expressed in a wide range of tissues during development. Nrf2 has been shown to be dispensable for growth and development in mice. Nrf2-deficient mice, however, are impaired in oxidative stress defense. Loss of Nrf1 function in mice results late gestational embryonic lethality. To determine whether Nrf1 and Nrf2 have overlapping functions during early development and in the oxidative stress response, mice were generated that are deficient in both Nrf1 and Nrf2. In contrast to the late embryonic lethality in Nrf1 mutants, compound Nrf1, Nrf2 mutants die early between embryonic days 9 and 10 and exhibit extensive apoptosis that is not observed in the single mutants. Loss of Nrf1 and Nrf2 leads to marked oxidative stress in cells that is indicated by elevated intracellular reactive oxygen species levels and cell death that is reversed by culturing under reduced oxygen tension or the addition of antioxidants. Compound mutant cells also show increased levels of p53 and induction of Noxa, a death effector p53 target gene, suggesting that cell death is potentially mediated by reactive oxygen species activation of p53. Moreover, expression of genes related to antioxidant defense is severely impaired in compound mutant cells compared with single mutant cells. Together, these findings indicate that the functions of Nrf1 and Nrf2 overlap during early development and to a large extent in regulating antioxidant gene expression in cells (Leung, 2003).

Knockout studies have shown that the transcription factor Nrf1 is essential for embryonic development. Nrf1 has been implicated to play a role in mediating activation of oxidative stress response genes through the antioxidant response element (ARE). Because of embryonic lethality in knockout mice, analysis of this function in the adult knockout mouse was not possible. Mice with somatic inactivation of nrf1 in the liver developed hepatic cancer. Before cancer development, mutant livers exhibit steatosis, apoptosis, necrosis, inflammation, and fibrosis. In addition, hepatocytes lacking Nrf1 showed oxidative stress, and gene expression analysis showed decreased expression of various ARE-containing genes, and up-regulation of CYP4A genes. These results suggest that reactive oxygen species generated from CYP4A-mediated fatty acid oxidation work synergistically with diminished expression of ARE-responsive genes to cause oxidative stress in mutant hepatocytes. Thus, Nrf1 has a protective function against oxidative stress and, potentially, a function in lipid homeostasis in the liver. Because the phenotype is similar to nonalcoholic steatohepatitis, these animals may prove useful as a model for investigating molecular mechanisms of nonalcoholic steatohepatitis and liver cancer (Xu, 2005).

The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. This study determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, a dominant-negative Nrf2 mutant (dnNrf2) was expressed in the epidermis of transgenic mice. The functionality of the transgene product was verified in vivo using mice doubly transgenic for dnNrf2 and an Nrf2-responsive reporter gene. Surprisingly, no abnormalities of the epidermis were observed in dnNrf2-transgenic mice, and even full-thickness skin wounds healed normally. However, the onset, incidence, and multiplicity of chemically induced skin papillomas were strikingly enhanced, whereas the progression to squamous cell carcinomas was unaltered. Evidence is provided that the enhanced tumorigenesis results from reduced basal expression of cytoprotective Nrf target genes, leading to accumulation of oxidative damage and reduced carcinogen detoxification. These results reveal a crucial role of Nrf-mediated gene expression in keratinocytes in the prevention of skin tumors and suggest that activation of Nrf2 in keratinocytes is a promising strategy to prevent carcinogenesis of this highly exposed organ (auf dem Keller, 2006).

Transcriptional targets of Erect wing homologs

Using genome-wide analysis of transcription factor occupancy, this study investigated the mechanisms underlying three mammalian growth arrest pathways that require the pRB tumor suppressor family. It was found that p130 and E2F4 cooperatively repress a common set of genes under each growth arrest condition and showed that growth arrest is achieved through repression of a core set of genes involved not only in cell cycle control but also mitochondrial biogenesis and metabolism. Motif-finding algorithms predicted the existence of nuclear respiratory factor-1 (NRF1) binding sites in E2F target promoters, and genome-wide factor binding analysis confirmed these predictions. NRF1, a factor known to regulate expression of genes involved in mitochondrial function, is a coregulator of a large number of E2F target genes. These studies provide insights into E2F regulatory circuitry, suggest how factor occupancy can predict the expression signature of a given target gene, and reveal pathways deregulated in human tumors (Cam, 2003).

In vertebrates, mitochondrial DNA (mtDNA) transcription is initiated bidirectionally from closely spaced promoters, HSP and LSP, within the D-loop regulatory region. Early studies demonstrated that mtDNA transcription requires mitochondrial RNA polymerase and Tfam, a DNA binding stimulatory factor that is required for mtDNA maintenance. Recently, mitochondrial transcription specificity factors (TFB1M and TFB2M), which markedly enhance mtDNA transcription in the presence of Tfam and mitochondrial RNA polymerase, have been identified in mammalian cells. This study establish that the expression of human TFB1M and TFB2M promoters is governed by nuclear respiratory factors (NRF-1 and NRF-2), key transcription factors implicated in mitochondrial biogenesis. In addition, NRF recognition sites within both TFB promoters are required for maximal trans activation by the PGC-1 family coactivators, PGC-1alpha and PRC. The physiological induction of these coactivators has been associated with the integration of NRFs and other transcription factors in a program of mitochondrial biogenesis. Finally, the TFB genes are up-regulated along with Tfam and either PGC-1alpha or PRC in cellular systems where mitochondrial biogenesis is induced. Moreover, ectopic expression of PGC-1alpha is sufficient to induce the coordinate expression of all three nucleus-encoded mitochondrial transcription factors along with nuclear and mitochondrial respiratory subunits. These results support the conclusion that the coordinate regulation of nucleus-encoded mitochondrial transcription factors by NRFs and PGC-1 family coactivators is essential to the control of mitochondrial biogenesis (Gleyzer, 2005).

Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by Nrf1 and Nrf2. tert-Butylhydroquinone (TBH) induces human GCLC via Nrf2-mediated trans activation of the antioxidant-responsive element (ARE). Interestingly, TBH also induces rat GCLC, but the rat GCLC promoter lacks ARE. This study examined the role of Nrf1 and Nrf2 in the transcriptional regulation of rat GCLC. The baseline and TBH-mediated increase in GCLC mRNA levels and rat GCLC promoter activity were lower in Nrf1 and Nrf2 null fibroblasts than in wild-type cells. The basal protein and mRNA levels and nuclear binding activities of c-Jun, c-Fos, p50, and p65 were lower in these null cells and exhibited a blunted response to TBH. Lower c-Jun and p65 expression also occurs in Nrf2 null livers. Levels of other AP-1 and NF-kappaB family members were either unaffected (i.e., JunB) or increased (i.e., Fra-1). Overexpression of Nrf1 and Nrf2 in respective cells restored the rat GCLC promoter activity and response to TBH but not if the AP-1 and NF-kappaB binding sites were mutated. Fra-1 overexpression lowered endogenous GCLC expression and rat GCLC promoter activity, while Fra-1 antisense had the opposite effects. In conclusion, Nrf1 and Nrf2 regulate rat GCLC promoter by modulating the expression of key AP-1 and NF-kappaB family members (Yang, 2005).

Post-translational modification of Erect wing homologs

Cyclin D1 promotes nuclear DNA synthesis through phosphorylation and inactivation of the pRb tumor suppressor. This study shows that cyclin D1 deficiency increases mitochondrial size and activity that is rescued by cyclin D1 in a Cdk-dependent manner. Nuclear respiratory factor 1 (NRF-1), which induces nuclear-encoded mitochondrial genes, is repressed in expression and activity by cyclin D1. Cyclin D1-dependent kinase phosphorylates NRF-1 at S47. Cyclin D1 abundance thus coordinates nuclear DNA synthesis and mitochondrial function (C. Wang, 2006).


REFERENCES

Search PubMed for articles about Drosophila erect wing

auf dem Keller, U., et al. (2006). Nrf transcription factors in keratinocytes are essential for skin tumor prevention but not for wound healing. Mol. Cell. Biol. 26(10): 3773-84. 16648473

Becker, T. S., et al. (1998). not really finished is crucial for development of the zebrafish outer retina and encodes a transcription factor highly homologous to human Nuclear Respiratory Factor-1 and avian Initiation Binding Repressor. Development 125(22): 4369-78. PubMed Citation: 9778497

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Chau, C. M., Evans, M. J. and Scarpulla, R. C. (1995). Nuclear respiratory factor 1 activation sites in genes encoding the gamma-subunit of ATP synthase, eukaryotic initiation factor 2 alpha, and tyrosine aminotransferase. Specific interaction of purified NRF-1 with multiple target genes. J. Biol. Chem. 267: 6999-7006. PubMed Citation: 1348057

Chen, L., et al. (2003). Nrf1 is critical for redox balance and survival of liver cells during development. Mol. Cell. Biol. 23(13): 4673-86. 12808106

de la Pompa, J. L., Garcia, J. R. and Ferrús, A., (1989). Genetic analysis of muscle development in Drosophila melanogaster. Dev. Biol. 131: 439-454. PubMed Citation: 2492244

deSimone, S. M. and White, K. (1993). The Drosophila erect wing gene, which is important for both neuronal and muscle development, encodes a protein which is similar to the sea urchin P3A2 DNA binding protein. Mol. Cell. Biol. 13: 3641-49. PubMed Citation: 8388540

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Fleming, R. J., DeSimone, S. M. and White, K. (1989), Molecular isolation and analysis of the erect wing locus in Drosophila melanogaster. Mol. Cell. Biol. 9: 719-725. 89219066

Gleyzer, N., Vercauteren, K. and Scarpulla, R. C. (2005). Control of mitochondrial transcription specificity factors (TFB1M and TFB2M) by nuclear respiratory factors (NRF-1 and NRF-2) and PGC-1 family coactivators. Mol. Cell. Biol. 25(4): 1354-66. 15684387

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Haussmann I., White, K. and Soller, M. (2008). Erect wing regulates synaptic growth in Drosophila by integration of multiple signaling pathways. Genome Biol. 9: R73. PubMed Citation: 18419806

Haussmann, I. U. and Soller, M. (2010). Differential activity of EWG transcription factor isoforms identifies a subset of differentially regulated genes important for synaptic growth regulation. Dev. Biol. 348(2): 224-30. PubMed Citation: 20854801

Hoog, C., et al. (1991). Gene regulatory factors of the sea urchin embryo. II. Two dissimilar proteins, P3A1 and P3A2, bind to the same target sites that are required for early territorial gene expression. Development 112: 351-64. PubMed ID: 1769340

Hsiao, H. Y., Jukam, D., Johnston, R., Desplan, C. (2013) The neuronal transcription factor erect wing regulates specification and maintenance of Drosophila R8 photoreceptor subtypes. Dev Biol 381: 482-490. PubMed ID: 23850772

Huo, L. and Scarpulla, R.C. (2001). Mitochondrial DNA instability and peri-implantation lethality associated with targeted disruption of nuclear respiratory factor 1 in mice. Mol. Cell. Biol. 21: 644-654. 11134350

Koushika, S. P., et al. (1999). Differential and inefficient splicing of a broadly expressed Drosophila erect wing transcript results in tissue-specific enrichment of the vital EWG protein isoform. Mol. Cell. Biol. 19: 3998-4007. PubMed ID: 10330140

Koushika, S. P., Soller, M. and White, K. (2000). The neuron-enriched splicing pattern of Drosophila erect wing is dependent on the presence of Elav protein. Mol. Cell. Biol. 1836-1845. PubMed ID: 10669758

Leung L., et al. (2003). Deficiency of the Nrf1 and Nrf2 transcription factors results in early embryonic lethality and severe oxidative stress. J. Biol. Chem. 278(48): 48021-9. 12968018

Rai, M., Katti, P. and Nongthomba, U. (2014). Drosophila Erect wing (Ewg) controls mitochondrial fusion during muscle growth and maintenance by regulation of the Opa1-like gene. J Cell Sci 127: 191-203. PubMed ID: 24198395

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Biological Overview

date revised: 10 February 2014

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